The disclosure relates generally to the field of a delivery vehicle for probiotic bacteria comprising a dry matrix of polysaccharides, saccharides and polyols in a glass form. Methods of making and uses thereof are also provided.
Probiotics are defined as live microbes that beneficially affect the host by modulating mucosal and systemic immunity, as well as improving intestinal function and microbial balance in the intestinal tract. Various nutritional and therapeutic effects have been ascribed to probiotics including: modulating immune response, lowering serum cholesterol concentrations, improving lactose intolerance symptoms, increasing resistance to infectious intestinal diseases, decreasing duration of diarrhea, reducing blood pressure, and helping to prevent colon cancer (Isolauri E et al. 2001, Kailasapathy K and J. 2000, Marteau P R et al. 2001, Perdigon G et al. 2001). In order to exert their beneficial effects on the host, probiotics must remain viable and reach the intestine in large numbers (Favaro-Trindade and Grosso 2002). However, maintaining long term stability of probiotics requires special storage conditions, since viability deteriorates rapidly over a short time period at ambient temperature and humid conditions (Shah 2000). In addition to poor shelf life, a significant loss of viability occurs upon exposure of the probiotics to gastric conditions of low pH and digestive enzymes. Existing preservation methods fail to provide satisfactory viability upon storage and gastric protection, especially if cells are stored at ambient or higher temperature and humidity.
Freeze-drying is often used for preservation and storage of bacteria because of the low temperature exposure during drying. However, it has the undesirable characteristics of significantly reducing viability as well as being time and energy-intensive. Freeze-drying involves placing the cells in solution, freezing the solution, and exposing the frozen solid to a vacuum under conditions wherein it remains solid and the water and any other volatile components are removed by sublimation. Standard freeze drying temperature of −30° C. to −70° C. are below the freezing point of water, but are well above the glass transition (Tg) temperature of the drying solution, which results in the undesirable effect of crystallization of water into ice. Freezing bacterial cultures results in substantial physical damage to the bacterial cell wall and subsequent loss of viability. Therefore, avoiding ice formation during cold storage of proteins, viruses, cells, tissues, and organs is an important problem in cryobiology.
The freezing point of water can be lowered by adding solutes that lower the vapor pressure of water. Freezing point depression is the physical basis on which essentially all currently used antifreeze agents (e.g., glycols, sugars and salts) perform. The disadvantage of freezing point depressors, known as cryoprotectants, is that large quantities of solutes (10% or more) are required to lower the freezing point by even a few degrees Celsius. At sufficiently high concentrations (typically 50% or more), conventional antifreeze agents can prevent ice formation, allowing aqueous solutions to be cooled to temperatures well below 0° C. without freezing. However, cryoprotectants are generally toxic at the high concentrations required to achieve glass formation or vitrification.
Other methods used to prepare dry and stable preparations of probiotics such as desiccation at ambient temperature and spray drying also has drawbacks. Desiccation at low or ambient temperature is slow, requires extra precautions to avoid contamination, and often yields unsatisfactory viability. Spray drying involves short excursions to relatively high processing temperatures and results in viability losses and limited storage times, even when stabilizing excipients are used (Lievense L C, van't Riet K. 1994).
A viable and stable formulation for intestinal targeting of probiotics has been described by Simmonds et al. (2005). The process requires the granulation of lyophilized bacteria with microcrystalline cellulose stabilizers such as skim milk, salts or short chain sugars and a disintegrant such as starch or alginic acid. The granulated semi dry bacteria are then desiccated at 40-70° C. to reduce the residual moisture level to less than 2 percent. This is followed by coating with an enteric agent and plasticizer. This multi-step process results in large particle size (over 425 micron) and still results in up to 1.5 logs loss of viability. An additional disadvantage of this method is the high content of the enteric coating agents (over 25% of the microsphere weight), which are mostly synthetic and not recognized as food grade materials. An inherent disadvantage of a coating procedure is that the relative proportion of the coating to active agent goes up by a cubic function of the particle, as the particle size gets smaller, making the process less usable for the production of particles of sizes less than 300 micron.
An alternative method of bacterial preservation has been described which uses a foam formation technique while eliminating the formation of ice crystals (Bronshtein et al. 2004, Roser et al. 2004). This method requires high concentrations of sugars (a combination of methylated mono, di- and oligo-saccharides) in the drying media and a freeze drier that is equipped with a controlled vacuum system and temperature exposure, and the addition of foam forming elements and stabilizers. In spite of some advantages of this method in achieving longer shelf life stability, the foam-preserved bacteria are not protected from gastric excursion. Furthermore this process is difficult and costly to scale up because the foam requires, by definition, large volumes of space under reduced atmospheric pressure (i.e., in a vacuum) for the production of very little mass. In addition, this material is very sensitive to humidity and the product will take up water readily, decreasing the viability of the bacteria.
A composition containing a sugar (trehalose) partly in amorphous glassy phase and partly in crystalline hydrate phase has been proposed by Franks et al (2003). The crystalline hydrate phase serves as an agent to dehydrate the amorphous phase, thereby enhancing the glass transition temperature of the amorphous glassy state. This composition was shown to stabilize single molecules such as proteins or nucleotides. The glass transition temperature of a mixture depends, among other factors, on its chemical composition (sugars, proteins, salts) and the moisture content, with water acting as a plasticizer, depressing the glass temperature. If, at any time, the glass transition temperature (Tg) is exceeded, either by exposure to heat or in consequence of moisture migration into the product, the amorphous glassy state may become liable to irreversible phase separation by crystallization. If crystallization occurs, any residual amorphous phase will then be composed of the other components and the moisture, resulting in a further depression of the glass transition temperature.
A glass is an amorphous solid state that is obtained by controlled desiccation of a solution. The advantage of the glassy phase in achieving long term stability results from the fact that diffusion in glassy (vitrified) materials occurs at extremely low rates (e.g., microns/year). Glassy materials normally appear as homogeneous, transparent, brittle solids, which can be ground or milled into a powder. The optimal benefits of vitrification for long-term storage are observed under conditions where Tg is greater than the storage temperature. The Tg is directly dependent on water activity and temperature, and may be modified by selecting an appropriate combination of solutes (i.e., polysaccharides, sugars, salts and proteins).
Glass formation occurs naturally in some plant and arthropod species that are very desiccation tolerant. A number of mosses and ferns, so-called resurrection plants, can undergo severe desiccation and survive for many years in a quiescent metabolic state only to revive upon the return of water to the environment. In most cases, the adaptation characteristic is to increase internal concentrations of certain saccharides such as trehalose to a level that form glassy states.
Prior to the current disclosure, no one has been able to provide a common and cost effective solution to the separate problems facing the probiotic industry, namely maintaining long shelf life stability {i.e., viability) of bacterial cells at ambient temperatures and high water activities (or high humidity) and providing gastric protection to minimize losses of probiotic viability during the transit through the stomach. The present invention overcomes these problems.